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Butea monosperma and chemomodulation: protective role against thioacetamide-mediated hepatic alterations in Wistar rats.


The present study was carried out to study the effect of Butea monosperma, a known liver acting drug on the tumor promotion related events of carcinogenesis in rat liver. Thioacetamide (TAA) was used to induce tumor promotion response and oxidative stress and caused significant depletion in the detoxification and antioxidant enzyme armory with concomitant elevation in malondialdehyde (MDA (1) (Monochrome Display Adapter) The first IBM PC monochrome video display standard for text. Due to its lack of graphics, MDA cards were often replaced with Hercules cards, which provided both text and graphics. See PC display modes and Hercules Graphics. ) formation, hydrogen peroxide ([H.sub.2][O.sub.2]) generation, ornithine decarboxylase (ODC) activity and unscheduled DNA synthesis. However, B. monosperma pretreatment pretreatment,
n the protocols required before beginning therapy, usually of a diagnostic nature; before treatment.

pretreatment estimate,
n See predetermination.
 at two different doses restored the levels of the above-said parameters (p<0.001) in a dose-dependent manner. The alcoholic extract of B. monosperma used in the present study seems to offer dose-dependent protection and maintain the structural integrity of hepatic cells. This was evident from the significant reduction in TAA-induced serum GOT, GPT GPT glutamic-pyruvic transaminase; see alanine transaminase.

glutamic-pyruvic transaminase


glutamic-pyruvic transaminase.
, Lactate dehydrogenase (LDH LDH -lactate dehydrogenase.

lactate dehydrogenase


lactic acid dehydrogenase; see lactate dehydrogenase.
) and [gamma]-Glutamyl transpeptidase activity (GGT GGT

?-glutamyl transferase.

GGT Gammaglutamyltransferase, see there
) activities (p<0.001). These investigations validate the use of B. monosperma in liver disorders by Ayurvedic physicians. Overall results indicate that the methanolic extract of B. monosperma possesses hepatoprotective effects and also it might suppress the promotion stage via inhibition of oxidative stress and polyamine polyamine /poly·am·ine/ (-am´en) any compound, e.g., spermine or spermidine, containing two or more amino groups.

 biosynthetic pathway.

[c] 2005 Elsevier GmbH. All rights reserved.

Keywords: Butea monosperma; TAA; Oxidative stress; Promotion


Liver diseases are considered as one of the serious health problems, as it is an important organ for the detoxification and deposition of endogenous and exogenous substances. Steroids, vaccines and antiviral drugs which have been employed as a therapy for liver diseases, have potential adverse effects especially when administered for long terms. Hence, hepatoprotective drugs from plant sources seem to be attractive alternative. Ayurveda is a traditional system of medicine being practiced in Indian subcontinent for over 5000 years. In Ayurveda, several herbal drugs have been prescribed as 'liver tonics' to reduce the toxicity due to ingested xenobiotics. It gives an elaborate account of the medicinal plants, their uses in herbal therapeutics, based upon traditional wisdom and knowledge. Some plants mentioned in Ayurveda are highly reputed for their potential benefits in the treatment of liver disorders (Singh and Handa, 1995). Among them Butea monosperma Lam. Kuntze (Fabaceae), commonly known as palash, is well-documented medicinal plant, which is used in Ayurvedic system for liver ailments.

In the literature B. monosperma is ascribed to have many medicinal properties. It has been used as tonic, astringent, aphrodisiac and diuretic. Its flowers are widely used in the treatment of hepatic disorders and viral hepatitis, diarrhoea and possess anti-implantation activity (Chopra et al., 1956). Roots of B. monosperma are reported to be useful in the treatment of filariasis filariasis: see elephantiasis. , night blindness, helminthiasis hel·min·thi·a·sis
n. pl. hel·min·thi·a·ses
A disease that is caused by infestation with parasitic worms.

helminthiasis, helminthosis
, piles, ulcers and tumors. Pippali rasayana, an Indian Ayurvedic drug, employs B. monosperma and is used in the management of giadiasis (Agarwal et al., 1997). The bark is reported to possess antitumor and antiulcer activities. The root bark is used as an aphrodisiac, analgesic and antihelmintic whereas the leaves possess antimicrobial property (Kasture et al., 2000).

B. monosperma flowers contain butin, butein and butrin, isobutrin, palasitrin, coreipsin and isocoreipsin, chalcones, and aurones (Gupta et al., 1970). Butrin (7,3',4'-trihydroxyflavanone-7,3'-diglucoside) and isobutrin (3,4,2',4'-tetra-hydroxy-chalcone-3,4'-diglucoside) (Fig. 1) are the well-known antihepatotoxic principles of B. monosperma (Wagner et al., 1986). For the present study, we prepared the methanolic extract of B. monosperma, which contain flavonoids flavonoids, common plant pigment compounds that act as antioxidants, enhance the effects of vitamin C, and strengthen connective tissue around capillaries.
, butrin and isobutrin as the major components. The presence of these compounds in the extract was confirmed by Co-TLC with the authentic sample under UV lamp (Wagner et al., 1986).

Since B. monosperma, a known liver acting drug, is capable of inhibiting several disorders, especially those that act through the generation of reactive oxygen species, the present work is designed to study for (1) its action to scientifically prove or disprove its therapeutic efficacy as hepatoprotective agent and (2) to evaluate its antioxidative and antiproliferating potential against thioacetamide (TAA)-induced early biomarkers of hepatic tumor promotion.


Materials and methods


Male albino rats of Wistar strains weighing 180-200 g were housed in groups of six in large spacious polypropylene cages. Lighting was regulated to provide equal hours of light and dark. Animals were obtained from the central animal house facility of Jamia Hamdard, New Delhi, India. The animals were acclimatized to standard laboratory conditions (25[+ or -]10[degrees]C, 50[+ or -]15% relative humidity) 1 week prior to the actual commencement of the experiment. They were provided with standard food pellets (Hindustan Lever Ltd., India) and tap water ad libitum.


[[.sup.3]H]-thymidine (82 Ci/mmol) and [[.sup.1-14]C]-ornithine were purchased from Amersham Corporation (UK). All other chemicals used were obtained from Sigma Chemical Co. (St.Louis, MO, USA).

Plant material extraction

The extraction procedure was followed as described by Wagner et al. (1986). Briefly, 500 g dried powdered parts of B. monosperma were extracted with methanol in a soxhlet for 20 h. Then by removing the solvent under reduced pressure in rotatory ro·ta·to·ry
1. Of, relating to, causing, or characterized by rotation.

2. Occurring or proceeding in alternation or succession.
 evaporator (Buchi Rotavapour, Switzerland), we obtained 145g of orange powder. The concentrated methanolic fraction obtained was stored at 4 [degrees]C and was dissolved in distilled water to make the required doses. For the screening of presence of antihepatotoxic compounds isobutrin and butrin, 100 g of the total methanol extract was partitioned three times between water and ethyl acetate. After ethyl acetate fraction removal, the remaining water phase was treated three times with n-butanol. The solvent was removed under reduced pressure to yield 2.7 g butanol bu·ta·nol  
Either of two butyl alcohols derived from butane and used as solvents and in organic synthesis.

[butan(e) + -ol1.
 fraction (yellow powder). To screen the presence of active principles, the butanol fraction was chromatographed on silica gel. Isobutrin gave an intense yellow spot in TLC TLC total lung capacity; thin-layer chromatography.

1. thin-layer chromatography

 (visible and UV fluorescent) while butrin did not show any fluorescence. For the quantitative assays of flavonoids, butrin and isobutrin were isolated by preparative TLC on silica gel (0.5 mm, Merck) which give an approximate yield 5 mg of isobutrin and 4 mg butrin from 24 mg butanol fraction. This corresponds to 20-83% isobutrin and 16.6% butrin in the methanolic extract (Wagner et al., 1986).

Experimental protocol

To study the effect of pretreatment of animals with B. monosperma on TAA-mediated hepatic oxidative stress, ornithine decarboxylase (ODC) activity and DNA synthesis, 30 male rats were randomly taken in five groups and application was done in the following manner:</p> <pre> Groups Treatment regimen I. Only saline (0.5 ml/animal) II.

TAA (6.6 mmol/kg body wt., ip) III. B. monosperma (100 mg/kg body wt.) + TAA (6.6 mmol/kg body wt., ip) IV. B. monosperma (200 mg/kg body wt.) + TAA (6.6 mmol/kg body wt., ip) V.

Only B. monosperma (200 mg/kg body wt.) </pre> <p>Biochemical assays

ODC activity was determined by the method of O'Brien et al. (1975).

Hepatic DNA synthesis estimation was done by the method of Athar and Iqbal (1998).

GSH GSH reduced glutathione.


reduced glutathione.
 content in liver was determined by the method of Jollow et al. (1974).

Glutathione peroxidase (GPx) activity was assayed by the method described by Athar and Iqbal (1998).

Glutathione Reductase activity was assayed by the method of Carlberg and Mannervick (1975).

Quinone quinone

Any member of a class of cyclic organic compounds comprising a six-membered unsaturated ring (see saturation) to which two oxygen atoms are bonded as carbonyl groups (−C=O; see functional group).
 Reductase reductase /re·duc·tase/ (-tas) a term used in the names of some of the oxidoreductases, usually specifically those catalyzing reactions important solely for reduction of a metabolite.  (QR) activity was assayed by the method of Benson et al. (1980).

Xanthine Oxidase activity was assayed by the method of Athar et al. (1996).

Glutathione S-transferase activity was assayed by the method of Habig et al. (1974).

Lipid perooxidation estimation was done by following the method of Wright et al. (1981).

Hydrogen peroxide ([H.sub.2][O.sub.2]) content was estimated by the method of Pick and Keisari (1981).

Superoxide dismutase (SOD) activity was assayed by the method of Stevens et al. (2000).

Lactate dehydrogenase (LDH) activity was estimated by the method of Kornberg (1955).

Serum oxalacetate and pyruvate pyruvate /py·ru·vate/ (pi´roo-vat) a salt, ester, or anion of pyruvic acid. Pyruvate is the end product of glycolysis and may be metabolized to lactate or to acetyl CoA.

 transaminase activity (SGOT SGOT serum glutamic-oxaloacetic transaminase; see aspartate transaminase.

Serum glutamic aminotransferase; an enzyme that catalyzes the transfer of the amino group from glutamic acid to oxaloacetic acid
 & SGPT SGPT serum glutamic-pyruvic transaminase; see alanine transaminase.

Serum pyruvate aminotransferase; an enzyme in serum and body tissues that catalyzes the transfer of amino acid groups from
) was determined by the method of Reitman and Frankel (1957).

[gamma]-Glutamyl transpeptidase activity (GGT) activity was determined by the method of Orlowski and Meister (1973).

Protein concentration in all samples was determined by the method of Lowry et al. (1951).

Statistical analysis

The level of significance between different groups was based on Dunnett's t-test followed by the analysis of variance test (ANOVA anova

see analysis of variance.

ANOVA Analysis of variance, see there


Fig. 2 depicts the effects of prophylactic treatment of B. monosperma on TAA-mediated depletion in reduced glutathione, glutathione reductase and enhancement of glutathione S-transferase in rat liver. TAA caused 62% depletion in reduced glutathione content, 51% reduction in glutathione reductase and 82% elevation in glutathione S-transferase as compared to only saline-treated control group. Prophylaxis with B. monosperma at lower (100 mg/kg body wt.) and higher (200 mg/kg body wt.) doses, shown in groups III and IV, caused 18-33% recovery in reduced glutathione, 8-47% recovery in glutathione reductase and 11-40% reduction in glutathione S-transferase activity.



Fig. 3 shows the effects of prophylaxis of B. monosperma on TAA-mediated depletion in hepatic GPx, QR and enhancement of xanthine oxidase activities in rats. TAA caused 35% reduction in GPx, 49% reduction in QR and 97% induction in xanthine oxidase on comparison with saline-treated control group. Prophylaxis with B. monosperma caused 5-26%, 11-40% and 24-43% recovery of GPx, QR and xanthine oxidase, respectively, at both the doses.

Fig. 4 shows that treatment with TAA induced [H.sub.2][O.sub.2] formation (74%), lipid peroxidation (145%) and depletion in SOD (59%) in rat liver whereas pretreatment with B. monosperma caused 21-45% and 32-51% reduction, respectively, in hydrogen peroxide formation and lipid peroxidation and 18-45% recovery of SOD activity at low and high doses, respectively.

The effect of pretreatment of animals with B. monosperma on TAA-mediated alterations in the activities of serum enzymes GOT, GPT, LDH and GGT are shown in Table 1. TAA treatment leads to about 118%, 172%, and 60% and 102% enhancement in serum GOT, GPT, LDH and GGT, respectively, as compared with saline-treated control. Pretreatment with B. monosperma resulted in the 12-38%, 15-42%, 6-24% and 14-43% reduction in the values of serum GOT, GPT, LDH and GGT, respectively, as compared with the TAA-treated control at lower (100 mg/kg body wt.) and higher (200 mg/kg body wt.) doses of B. monosperma extract.


Table 2 depicts the effect of B. monosperma in the attenuation of TAA-induced ODC activity and DNA synthesis. The activity of ODC and the synthesis of DNA DNA: see nucleic acid.
 or deoxyribonucleic acid

One of two types of nucleic acid (the other is RNA); a complex organic compound found in all living cells and many viruses. It is the chemical substance of genes.
 increased to 549% and 957% in TAA-treated group. Lastly, pretreatment of B. monosperma caused 66% and 60% decline in the elevated ODC activity and 35-57% inhibition in case of DNA synthesis, respectively.


Natural compounds that reduce the chemical activating enzymes might be good candidates for protecting against chemically induced toxicities. Disturbance of normal prooxidant-antioxidant equilibrium in body leads to oxidative stress, which is characterized by the increased generation of reactive oxygen species. Reactive oxygen species play an important role in many human degenerative diseases including cancer (Sun, 1990). TAA is bioactivated by CYP CYP

In currencies, this is the abbreviation for the Cyprus Pound.

The currency market, also known as the Foreign Exchange market, is the largest financial market in the world, with a daily average volume of over US $1 trillion.
450 and/or flavin containing monooxygenase systems to toxic metabolites TAA sulfine (sulphoxide) and sulfene (sulphone Sul´phone

n. 1. (Chem.) Any one of a series of compounds analogous to the ketones, and consisting of the sulphuryl group united with two hydrocarbon radicals; as, dimethyl sulphone, (CH ) s>.SO .
), which produce centrilobular hepatic necrosis and apoptosis (Dogru-Abbasoglu et al., 2001).

In the present study, we have shown the effect of pretreatment of B. monosperma prior to TAA treatment at two doses and the results suggest that it may contribute to the chemopreventive effect. B. monosperma showed a significant recovery in the level of glutathione and its metabolizing enzyme in the liver induced the detoxifying enzyme system, which is shown by the elevated levels of other QR, SOD, GPx, and xanthine oxidase, which are important phase II enzymes. Lipid peroxidation is accepted to be one of the principal causes of chemically induced liver injury and is mediated by the production of free radicals. Pretreatment of animals with B. monosperma dose dependently decreased the level of TAA-induced lipid peroxides and [H.sub.2][O.sub.2] content. According to the result obtained, it can be said that B. monosperma exercised powerful free radical scavenging activity and may therefore act by scavenging the reactive oxygen species formed during the TAA metabolism. Any compound, natural or synthetic, with antioxidant properties that might contribute towards the partial or total alleviation of this damage, might have a significant role in maintaining health when used as a medicine or consumed as a part of the normal diet.

Damage to the structural integrity of liver is reflected by an increase in the level of serum transaminases because these are cytoplasmic in location and are released into circulation after cellular damage (Recknagel et al., 1991). The alcoholic extract of B. monosperma used in the present study seems to offer dose-dependent protection and maintain the structural integrity of hepatic cells. This was evident from the significant reduction in TAA-induced enhancement in serum GOT, GPT, LDH and GGT activities. These investigations validate the use of B. monosperma in liver disorders by Ayurvedic physicians.

Oxidative stress, ODC induction and enhanced DNA synthesis are considered as early events of carcinogenesis. In various models of chemical carcinogenesis, carcinogen induces ODC activity and inhibitors of ODC suppress cancer development. ODC activity and [[.sup.3]H]-thymidine incorporation are used extensively as a biochemical marker to evaluate the tumor promoting potential of an agent. B. monosperma inhibits ODC activity. A significant decrease in the level of ODC activity and [[.sup.3]H]-thymidine incorporation by the pretreatment of B. monosperma was observed in a dose-dependent manner, which suggests its role as an antitumor promoting agent (Table 2).

Various constituents of B. monosperma include butrin, isobutrin, butin, butein and palasitrin, coreipsin and isocoreipsin, chalcones, and aurones. Combined activities of these constituents might be responsible for the antioxidative and antiproliferating activities of B. monosperma. Our data, however, provide a substantial amount of mechanistic approach to show the chemopreventive effect of B. monosperma against oxidative stress, and tumor promotion related aspects of carcinogenicity. Hence, the present study clearly demonstrates the role of B. monosperma in the inhibition of biochemical events of tumor promotion.


Dr. Sarwat Sultana is thankful to Indian Council of Medical Research (ICMR), New Delhi, India, for providing necessary funds to conduct this study.


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A. Sehrawat, T.H. Khan, L. Prasad, S. Sultana*

Section of Chemoprevention che·mo·pre·ven·tion
The use of chemical agents, drugs, or food supplements to prevent disease.

 and Nutrition Toxicology, Department of Medical Elementology and Toxicology, Faculty of Science, Jamia Hamdard (Hamdard University), New Delhi 110 062, India

Received 7 September 2004; accepted 10 November 2004

*Corresponding author. Tel.: +91 11 26089688; fax: +91 11 26059663.

E-mail address: (S. Sultana).
Table 1. Effect of pretreatment of animals with Butea monosperma on TAA-
mediated alterations in activities of serum oxaloacetate and pyruvate
transaminases (SGOT & SGPT), Lactate dehydrogenase (LDH) and [gamma]-
Glutamyl transpeptidase (GGT)

Treatment groups        SGOT (IU/l)             SGPT (IU/l)

Saline-treated control   90.79[+ or -]2.66       62.18[+ or -]3.22
TAA alone (6.6 mmol/kg  197.6[+ or -]2.09*      169.1[+ or -]0.57*
in 0.9% NaCl)
B. monosperma (100 mg/  173.3[+ or -]4.06 (##)  144.06[+ or -]4.64 (#)
kg body wt.) + TAA
(6.6 mmol/kg body wt.)
B. monosperma (200 mg/  123.0[+ or -]9.79 (##)   97.10[+ or -]4.17 (##)
kg body wt.) + TAA
(6.6 mmol/kg body wt.)
B. monosperma only       86.71[+ or -]3.80       54.12[+ or -]2.23
(200 mg/kg body wt.)

                        LDH (nmol NADH           GGT (nmol p-
                        oxidized/min/mg          nitroaniline formed
Treatment groups        protein)                 min/mg/protein)

Saline-treated control  455.7[+ or -]13.28       440.2[+ or -]25.14
TAA alone (6.6 mmol/kg  728.2[+ or -]16.54*      944.2[+ or -]24.35*
in 0.9% NaCl)
B. monosperma (100 mg/  683.9[+ or -]5.39 (#)    809.0[+ or -]44.24 (#)
kg body wt.) + TAA
(6.6 mmol/kg body wt.)
B. monosperma (200 mg/  549.9[+ or -]17.31 (##)  539.3[+ or -]17.81(##)
kg body wt.) + TAA
(6.6 mmol/kg body wt.)
B. monosperma only      435.6[+ or -]8.24        440.2[+ or -]25.14
(200 mg/kg body wt.)

Each value represents means [+ or -] S.E.; n = 6. *p < 0.001 compared to
corresponding value for saline-treated control. (#) p < 0.05 and (##) p
< 0.001 compared with the corresponding value for treatment with

Table 2. Effect of pretreatment of Butea monosperma in the attenuation
of TAA-induced hepatic ornithine decarboxylase (ODC) activity and DNA

                                                 DNA synthesis
                        ODC activity             [[.sup.3]H]-thymidine
                        (pmo[.sup.14]C[O.sub.2]  incorporation dpm/
Treatment groups        released/hr/mg protein)  [micro]g DNA

Saline-treated control  1.65[+ or -]0.031        296.69[+ or -]39.77
TAA alone (6.6 mmol/kg  4.07[+ or -]0.059*       682.64[+ or -]31.40*
in 0.9% NaCl)
B. monosperma (100 mg/  2.85[+ or -]0.066 (#)    472.37[+ or -]15.65 (#)
kg body wt.) + TAA
(6.6 mmol/kg body wt.)
B. monosperma (200 mg/  1.92[+ or -]0.045 (#)    388.95[+ or -]30.54 (#)
kg body wt.) + TAA
(6.6 mmol/kg body wt.)
B. monosperma only      1.59[+ or -]0.048        224.12[+ or -]27.02
(200 mg/kg body wt.)

Each value represents means [+ or -] S.E.; n = 6. *p < 0.001 compared to
corresponding value for saline-treated control. (#) p < 0.001 compared
with the corresponding value for treatment with thioacetamide.
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Author:Sehrawat, A.; Khan, T.H.; Prasad, L.; Sultana, S.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Geographic Code:1USA
Date:Feb 1, 2006
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